Influence of DOM and its subfractions on the mobilization of heavy metals in rhizosphere soil solution

Long-term industrial pollution, wastewater irrigation, and fertilizer application are known factors that can contribute to the contamination of heavy metals (HMs) in agricultural soil. In addition, dissolved organic matter (DOM) plays key roles in the migration and fate of HMs in soil. This study investigated the effects of amending exogenous DOM extracted from chicken manure (DOMc), humus soil (DOMs), rice husk (DOMr), and its sub-fractions on the mobilization and bio-uptake of Cd, Zn, and Pb. The results suggested that the exogenous DOM facilitate the dissolution of HMs in rhizosphere soil, and the maximum solubility of Zn, Cd, and Pb were 1264.5, 121.3, and 215.7 μg L−1, respectively. Moreover, the proportion of Zn-DOM and Cd-DOM increased as the DOM concentration increased, and the highest proportions were 97.5% and 86.9%. However, the proportion of Pb-DOM was stable at > 99% in all treatments. In addition, the proportion of hydrophilic acid (Hy) and Pb/Cd in the rhizosphere soil solution were 17.5% and 8.3%, respectively. This finding suggested that the Hy-metals complex has a vital influence on the mobilization of metals, besides its complexation with fulvic acid and humic acid. Furthermore, the elevated DOM addition contributed to an increment of HMs uptake by Sedum alfredii, in the following order, DOMc > DOMs > DOMr. This study can provide valuable insights to enhance the development of phytoremediation technologies and farmland manipulation. Since the risk that exogenous DOM would increase the uptake of HMs by crops, it is also needed to evaluate this case from an agricultural management perspective.

Widespread use of chemical fertilizers and wastewater irrigation is affecting agriculture soils due to contamination of HMs (e.g. Cd, Pb, Zn and Ni) 1 . Therefore, Cd and Pb are common non-essential and poisonous elements in contaminated soils, while Zn is an essential plant nutrient which is a trace element in plants 2 . However, excessive Zn concentrations can negatively affect the metabolism of microorganisms and growth of plants. Moreover, the migration and fate of HMs in soil is dependent on their chemical speciation and influenced by various environmental conditions such as soil agrotypes, soil pH, and dissolved organic matter (DOM) 3 . In fact, DOM, as a highly active material in terrestrial and aquatic ecosystems, represents the mobile phase of organic matter 4,5 . Previous studies suggested that DOM is a heterogeneous mixture and metals can complex with the fulvic acid (FA), humic acid (HA), and other hydrophilic acid by their phenolic hydroxyl, carboxyl and carbonyl groups 6,7 .
Regardless of the source, DOM can either enhance or inhibit the mobility of metals in soils, depending on its origin or specific properties 8 . On one hand, the macromolecular weight, complexation and precipitation with heavy metals of the organic matter can suppress the bioavailability of metals. On the other hand, the low molecular weight DOM can dissolve the metals with the formation of soluble metal-organic complexes, enhancing the metal bioavailability in soils 9 . Consequently, the abundance of the active groups and the dominating composition of DOM predominantly modify the combination with trace metals and hence influence their speciation and bioavailability. Previous studies mainly focused on the effect of endogenous DOM on the speciation of HMs in arable soil, but studies about exogenous DOM have been ignored.
Pot experiment. Each subsample of 1.0 kg contaminated soil was placed in plastic pots and amended with four levels of each DOM type at 25, 50, 100, and 200 mgC kg −1 soil (denoted as T25, T50, T100, T200, respectively). Sufficient amounts of NH 4 NO 3 were used as a supplement to balance the supply of N. The hyperaccumulating ecotypes of S. alfredii of similar sizes were then transplanted into the pots and each treatment was done in quadruplicate. Pot soils with no DOM addition were included as a control (T0). The deionized water was added to keep the water holding capacity approximately at a maximum of 65% by watering every 3-7 days 16 . The plants grew in a greenhouse with controlled light and temperature conditions (20-26 °C day/night temperature) 16 , and were harvested 90 days after the planting.
At the end of the experiment, each plant was separated into root and shoot and oven dried for 48 h at 65 °C. Finally, the rhizosphere soil (i.e. soil tightly adhering to the roots) were collected. The trace metals in plant samples were determined by atomic absorption spectrometry (AAS) after digestion with a mixture of 6 mL HNO 3 and 4 mL HClO 4 14 . A certified soil reference material (GBW07401) and blank treatments were used for quality control.
DOM extraction and fractionation. First, the selected materials of rice hull and chicken manure were air-dried and ground. Samples, including the soil, were mixed with deionized-distilled water with a ratio of 1:20 (w/v) and kept at 25 ± 1 °C for fermentation purposes for a month. Then, the mixture was shaken (200 rpm) for 24 h at 25 °C and centrifuged at 10,000 × g for 20 min 8 . Subsequently, the supernatant was filtered through 0.45 μm microfiltration membranes, and the exogenous DOM was obtained.
DOM was fractionated into three sub-fractions including fulvic (FA) and humic acid (HA), hydrophilic acid (Hy), and hydrophobic neutral organic matter (HON) 17 . At the beginning of experiment, about 25 mL of the sample solution was adjusted to pH 1 with HCl (6 M) to precipitate the dissolved HA 16 . After, the DOC concentration of the pellet with the precipitated HA was measured by removing the supernatant and re-dissolved in 5 mL of 0.1 M KOH 16 . The XAD-8 resin (Sigma) was previously prepared and washed successively with 0.1 M HCl and NaOH solutions, Soxhlet extractions with methanol, and ultrapure water. The clean resin (~ 4 g) was added into the remaining supernatant (representing the dissolved FA + Hy + HON) to adsorb the FA and HON fractions. The suspension was then filtered through a 0.45 μm filter after 1 h of settling by continuous shaking, www.nature.com/scientificreports/ and a subsample of the filtrate was taken to measure the DOC in the Hy fraction. Additionally, the resin with 10 mL of 0.1 M KOH was equilibrated for 1 h to desorb FA, and the suspension was taken for DOC analysis. Finally, the HON fraction on the resin was quantified as the difference between the total amount of the adsorbed (FA + HON) and the amount of desorbed FA 16,18 . During the experiment, the control was also used to examine the DOC contribution from DAX-8 resin. All the subsamples were analyzed using a total organic carbon (TOC) analyzer (Analytik Jena Multi N/C 3100).

Soil solution analysis.
Each pot was saturated with Milli-Q water and equilibrated for 18 h prior to plant harvesting. Then, the soaking soil was packed into 25-mL filtration tubes and centrifuged at 8000 g for 30 min. The filtration tube was put in a 50-mL centrifuge tube which contained a small spacer in the bottom. The extracted solution was centrifuged (12,000 g, 30 min) again and passed through a 0.45 μm membrane filter. The total dissolved K, Ca, Na, Mg, and anions (SO ), and total dissolved K, Ca, Na, Mg, Cd, Pb, and Zn in soil solution were evaluated 18 . The NICA-Donnan model in Visual MINTEQ was selected to evaluate the metal binding to DOM and calculated using the proportions of HA and FA in the three exogenous DOM according to a previous study 19 .
The exogenous DOMs and the T100 treatment (100 mgC kg −1 soil) were considered to further evaluate the influence of DOM sub-fractions on the complexation with metals. Also, the sub-fraction of hydrophilic acid, along with the measured FA and HA fractions, were used to calculate the Cd, Pb, and Zn binding to organic matter. Since the low molecular weight organic acid (LMWOA) is an important constituent of the hydrophilic acid and apt to complex with heavy metals, its parameters were considered in the NICA-Donnan model. Moreover, the LMWOA can account for 30% of hydrophilic acid, including 70% of single carboxylic acid, 20% of dicarboxylic acid, and 10% of tricarboxylic acid (represented as acetic acid, oxalic acid and citric acid, respectively) 20 Table S1. The concentration of DOC in the solution increased with the DOM concentration (T200 > T100 > T50 > T25 > T0) ( Fig. 1). In fact, the maximum DOM concentrations were 254.2 mg L −1 , 261.9 mg L −1 , and 284.2 mg L −1 for DOMr, DOMs, and DOMc, respectively ( Fig. 1). Overall, the DOM concentration in the rhizosphere soil solution was higher than that of bulk soil with the same treatment, which could be attributed to the root excretion and microbial activities in the rhizosphere area. Moreover, the addition of DOMc resulted in the highest amount of DOC and dissolved Cd, Pb, and Zn, whereas the addition of DOMr yielded a lower amount compared to the other two exogenous DOM. Furthermore, the total dissolved heavy metals raised along with the increasing DOM amount. The concentrations of Cd, Pb, and Zn in the rhizosphere soil solution were 42.5-121.3 μg L −1 , 65.4-215.7 μg L −1 and 446.7-1264.5 μg L −1 , respectively (Fig. 1).
These findings confirmed that the abundance of active functional groups and structural heterogeneity of DOM can influence the dissolution of heavy metals in the soil solution. In fact, it was reported before that DOMc contains strong active functional groups such as -COOH, C=C, and C=O, which could act as potential chelating centers and facilitate the solubility of metals 19 . Also, Kim et al. 22 showed that the addition of DOM to soil can lead to decreased S/L (soil solid phase/liquid phase) ratios, desorb metals from soil surface, and promote the dissolution of metals in soil solution. Moreover, the present study showed that the dissolved metals in soil solution increased with the DOM increasing amounts, which agreed with previous studies. Other studies also found that the increasing concentrations of DOC after dairy-manure amendment triggered the dissolution of metals, and there was a further significant positive correlation between the concentration of metals and DOC in the soil solution 5,23 . In this study, the content of Cd and Zn in soil solution was higher than Pb. This finding was consistent with the pK hydrolysis values of Pb 2+ , Cd 2+ and Zn 2+ (7.7, 10.08 and 8.96, respectively) because the small hydrolysis constant was usually accompanied with the lower solubility and bioavailability of metals 24 . In addition, the lower solubility of Pb could also be caused by its strong affinity to soil carbonate or Fe/Mn minerals that could limit the release from the solid phase 25 . Moreover, the dynamic equilibrium between the mobilization and replenishment of heavy metals in soil and the uptake by plants could explain the difference of metal concentrations of the rhizosphere and bulk solutions 26 . In this study, the reduction of metals in the rhizosphere soil solution could be related to the higher efficiency of root absorption than that in the replenishment of dissolved metals.  (Tables S2-S3). Compared to the free ions, the proportions of organic complex Cd and Zn in the rhizosphere soil solution were higher, and these proportions increased along with the DOM addition treatments. Moreover, the highest proportions of organic Cd and Zn complexes in solution were 97.5% (DOMr treatment) and 86.9% (DOMc treatment), respectively. In addition, the proportions of Zn 2+ in the soil solution were higher than those of Cd 2+ in the same treatment. For example, the proportion of Zn 2+ in different treatments ranged from 33.2% (T200) to 70.6% (T25), while Cd 2+ ranged from 2.2% (T200) to 11.9% in DOMr. In addition, the proportion of free Pb 2+ was less than 1%, and the organic complex Pb was more than 99% for all treatments (Table S4). There was no inorganic complex Pb species in the soil solution.
These results were consistent with other previous investigations. For instance, Li et al., found that the affinity of Pb with soluble organic ligands in soil solution was significantly stronger than that of Cd and Zn. Therefore, the proportion of organic-Pb was higher 8 . Brümmer investigated the complexation of Cd and Pb with humic acids and demonstrated that the affinity of Pb with humic acids was much higher than that of Cd, with more stable and heterogeneous complexes 27 . The distinct speciation of the three metals in soil solution could be attributed to the differences in chemical characteristics and the ability to form different organic complexes. In addition, Essington indicated that the distribution of electron layers, the ability of accepting electron pairs, and the complexing ability of the same ligand with metal ions is significantly different 24 . In fact, the complexation process of metal ions with organic ligands depends on the stability constant (logK f ), with the order of  T0 T25 T50 T100 T200 T25 T50 T100 T200 T25 T50 T100 T200   0  www.nature.com/scientificreports/ Cu 2+ > Ni 2+ > Pb 2+ > Co 2+ > Zn 2+ > Cd 2+ > Fe 2+ > Mn 2+ . Therefore, the higher stability constant resulted in more proportion of metal-DOM and accelerated the metal desorbing from the soil solid particles 24,28 .
As concluded from other studies, the NICA-Donnan model can be a reasonable option to predict the metal speciation in the soil solution from wide range of origins, especially for Cd and Zn 18 . However, there is a need to update the generic binding parameters in the model for calculating the Pb speciation. Regarding the Pb speciation, the surface complexation and mineral precipitation/dissolution in the soil should be considered as well. Therefore, the Pb calculation data need a further analysis.

Effect of the DOM sub-fractions on metal speciation. Sub-fractions of DOM in the soil solution and
the proportion of different fractions complexation with metals are shown in Tables 2 and 3. The concentration of DOMs (T100) was 119.4 mg L −1 and 149.5 mg L −1 in bulk and rhizosphere soil solution, respectively, and it was mainly composed of Hy and FA fractions in the rhizosphere, accounting for 51.5% and 26.4%, respectively. Moreover, the proportions of HA in the bulk solution and HON in the rhizosphere solution were the lowest, with both proportions less than 10%. The NICA-Donnan model calculation indicated that the proportions of the total organic-Cd complex were 83.2%, 8.5%, and 8.3% for FA-Cd, HA-Cd, and Hy-Cd, respectively, while the HA-Pb was the dominant speciation complex followed by FA-Pb and Hy-Pb. There was no speciation detected in the Hy-Zn complex. Therefore, only the HA and FA data were used in the calculation.
In general, studies of DOM-metal complexes in soil solution and natural water mainly focused on the fulvic and humic acids. However, the other hydrophilic acids accounted for one third of DOM in most cases, and the contribution of its complexation ability to heavy metal ions in rhizosphere soil are seldom reported 29 . The findings of this study were different from other previous reports because the effect of LMWOA and other hydrophilic    17,19 . In fact, this study detected more hydrophilic substances in the rhizosphere soil solution due to the root exudation and the contribution of this fraction, especially LMWOA, to the Cd and Pb complexes. In fact, Tao et al. 12 indicated that the oxalic, malic, and tartaric acids were the predominant LMWOA in the rhizosphere soil solution of S. alfredii. Moreover, the tartaric acid was identified as the unique root exudate, which was mainly distributed within the 0-6 mm rhizosphere range. In fact, the exudation of tartaric acid of LMWOA was highly efficient in the Cd solubilization due to the formation of soluble Cd-tartrate complexes 11 . Despite the low aromaticity of the hydrophilic part, its concentration must be considered when the concentration of active humus is used as an input in the model calculation. Furthermore, the ability of the active humus to complex with heavy metals still needs further investigation 29,30 .  T0 T25 T50 T100 T200 T25 T50 T100 T200 T25 T50 T100 T200 T0 T25 T50 T100 T200 T25 T50 T100 T200 T25 T50 T100 T200  www.nature.com/scientificreports/ respectively. In addition, concentrations of Cd and Zn in the shoot of S. alfredii were higher than those in the root, while the concentration of Pb was lower in the shoot than in the root. Yang et al. 13 reported that when Pb enters the plant root, it has an intercellular neutral pH, high phosphate, and high carbonate environment, so the transport of Pb from root to shoot is usually low. However, the maximum Pb content in the shoot and root of S. alfredii can reach to 514 and 13,922 mg kg −1 , respectively, when the Pb level was 320 mg L −1 31 . Furthermore, it was reported that the shoot of S. alfredii could uptake up to 5000 mg kg −1 Zn and 1400 mg kg −1 Cd at pot or field scale 13,32 . Also, the remediating efficiency of S. alfredii can vary under different circumstances 33 . The correlation among pH, different types and concentrations of DOM, dissolved metals in soil solution and the uptake HMs by root/shoot of S. alfredii were calculated to determine the influence of the DOM addition on the bioavailability of HMs (Fig. 4). A significant positive correlation was observed between DOM and the dissolved HMs and uptake by S. alfredii (P < 0.01), indicating that the three exogenous DOM can play a key role in the availability of HMs in soil. These observed correlations can be related to the high DOM concentration which can lead to the formation of HMs-DOM complexes and influence the transport and bioavailability of metals afterwards 34 . These findings also revealed that the correlation coefficient of DOM with the Cd and Zn concentrations in the root of S. alfredii was weaker than that of Pb, which agreed with the aforementioned results and discussion. Therefore, Cd and Zn could be accumulated in the shoot of S. alfredii, while the transfer ability of Pb from root to shoot was small. However, it was not possible to conduct an analysis of the correlation of the HMs speciation and bioavailability due to the limitation of the model in calculating the complexation of Pb-DOM, which needs a further study. Furthermore, although the soil pH did not have an apparent change, a significant negative correlation was observed between pH and the other factors (P < 0.05), suggesting that the increased pH was not conductive to the solubility and bioavailability of HMs. ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** * ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** www.nature.com/scientificreports/ The uptake of heavy metals by plants is influenced by various factors, such as the ecotype of the plant, speciation of metals, and the physicochemical properties of soil. In fact, Krishnamurti and Naidu proposed that the increase of organic matter content in soil after sludge application enhanced the proportion of Cd-DOM complex and the bioavailability of Cd 35 . This enhancement can further promoted the uptake of Cd by plants, which agree with the results of the present study. Moreover, Mao et al. denoted that the FA and some of the HA from compost played regulatory roles in enhancing lateral roots and root hairs and tips, which ameliorated the plant growth and constantly activated the Cd uptake by S. alfredii (285.09-709.72 µg plant −1 ) 36 . Also, Fitz and Wenzel demonstrated that hyperaccumulators can enhance the solubility of metals in the rhizosphere soil by root exudation or microbial activity, further promoting the absorption of metals by plants 37 . For example, the intake of exogenous DOM promoted the microbial diversity and community and increased the bioavailability of metals in soil under wetted conditions 38 . In addition, soil DOM can provide nutrient to the microorganisms, and the DOC concentration was positively correlated with the microbial activity. It is well known that soil microbes can affect the mobility and bioavailability of heavy metals by solubilizing metal-phosphates, releasing chelating agents, originating redox changes and through acidification events 39 .

Effect of DOM
Moreover, the exogenous DOM applied in the HMs-contaminated farmland could mobilize metals which can potentially been taken up by crops or even can be ended up in receiving waters such as rivers and draining streams in agricultural lands. As the uptake and transfer mechanisms of heavy metals by different types of crops are diverse, it is necessary to conduct in-depth research on the absorption of heavy metals and their risk assessment in the future. In addition, the concern of exogenous organic residuals, such as the rise hulls and chicken manure application in agricultural soil, should be addressed.

Conclusions
In this study, it was demonstrated that the addition of exogenous DOM promoted the dissolution of heavy metals in the rhizosphere soil, and the solubility of Cd and Zn was greater than that of Pb. Also, the proportion of organic complex speciations of Zn and Cd increased significantly with DOM concentrations, and the proportion of Zn 2+ was higher than Cd 2+ at the same DOM concentration. Furthermore, the proportion of DOM-Pb in the rhizosphere soil solution was over 99% in all treatments. The complexation of hydrophilic acid with Pb and Cd had an important effect on the mobilization and migration of these two metals, besides the complexation with fulvic acid and humic acid. Furthermore, the significant increasing order of Cd, Pb, and Zn concentrations (except Zn at T 25 ) in the shoot of S. alfredii were DOM C > DOMs > DOMr. These findings improved the current knowledge about the effects of exogenous DOM on the transformation of trace metals in the rhizosphere soil. Moreover, the application of exogenous DOM could be a useful alternative to improve the efficiency of phytoremediation. However, it is necessary to consider the risk that exogenous DOM can increase the uptake of HMs by crops from an agricultural management perspective. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.